Abstract: In eukaryotes the ATP dependent protein degradation by the ubiquitin-proteasome pathway removes short lived signaling protein that is critical in regulation of cellular process, degrades misfolded and damaged proteins whose accumulation is toxic to the cell and breaks down foreign proteins to generate antigenic peptides for presenting to the immune system. It is fundamental in understanding the mechanism of many human diseases, especially cancer and neurodegenerative diseases, e.g. Huntington disease. The eukaryotic 26S proteasome is formed by a 20S proteasome with the proteolytic active sites sequestered inside it and two 19S regulatory particles each contain six ATPases in contact with the 20S. A key role of the ATPases is to open the gated channel in the 20S to facilitate substrates enter for destruction. Because of the large size and dynamic nature of the 19S regulatory particle, crystallization of the entire 26S proteasome for structure determination remains unsuccessful despite substantial efforts, and the mechanism by which the ATPases controls the gate-opening in the 20S remains to be elucidated. We use an alternative structure determination technique to elucidate this mechanism: single particle electron cryomicroscopy (cryoEM) which does not require crystallization of proteasomal ATPases-20S complex. In collaboration with Professor Alfred Goldberg from Harvard Medical School, we have found that the ATPases only require their C-termini to induce the gate-opening. We thus separated the mechanistic studies of ATPase induced gate-opening from the structure determination of the ATPases. This application focuses on two critical issues of the proteasomal ATPases: (1) how the ATPases opens the gate in 20S and (2) the conformational changes of ATPases during the ATPase cycle. Our aims are clearly defined and our approach is novel, unique and has been proven successful. We already made a critical step forward by determining that the C-termini of ATPases induce a conformational change in the archaeal 20S that leads to its gate-opening. In Aim 1 we will explore the determinants that govern such conformational changes in archaeal 20S. In Aim 2, we will determine if the C-termini of eukaryotic 19S ATPases trigger similar conformational changes that lead to gate-opening in the eukaryotic 20S. In Aim 3 we will seek to elucidate the conformational changes of full length proteasomal ATPases during its ATPase cycle. Substantial completion of these aims will advance our knowledge about the proteasome-mediated protein degradation that plays a key role in the pathogenesis of many human diseases. It will also advance the technology of single particle cryoEM to achieve higher resolutions and to detect small ligand that is only a few residues in size.